Functional hyperemia is a hallmark of the healthy brain, but the underlying mechanisms remain elusive despite decades of research. It has recently emerged that functional hyperemia is initiated in the smallest blood vessels in the brain, capillaries: Our and other groups? work shows that activity-driven decreases in PO2 and increases in extracellular K+ ([K+]e), can directly increase the velocity of red blood cells (RBCs) passage through capillaries. We here propose to dissect the molecular pathways by which activity-induced increases in [K+]e and PO2 may induce capillary hyperemia and to ascertain if defects in BBB transport of K+ and PO2-induced RBC responses contribute to the suppression of neurovascular coupling in hypertension and aging. We will use microscopy with Gradient index (Grin) lenses to directly compare cortical and subcortical white matter capillary hyperemia and assess myelin damage, enabling direct cross-correlation of hypertension, aging, amplitude of hyperemia, PO2, K+ with white matter changes. The proposal will address these questions in a tightly linked set of in vivo and ex vivo experiments: Aim 1 will assess in vivo the hypothesis that activity-induced increases in parenchymal [K+]e are linked to active K+ transport across the BBB, resulting in elevated plasma [K+]. We will address capillary hyperemia in gray and white matter, where capillaries differ with regard to organization, density, and diameter. Aim 2 will use ex vivo analysis to test the hypothesis that elevated [K+]e activates the Na+/K+ ATPase in RBCs, leading to a decreased RBC volume and increased RBC velocity in the microfluidic chamber. In contrast, dips of PO2 drive capillary hyperemia by weakening the interactions between the RBC bilayer membrane and cytoskeleton, resulting in increased capillary velocity due to greater RBC membrane deformability. We further hypothesize that these two proposed mechanisms contribute differently to cortical and subcortical white matter capillary hyperemia. Aim 3 will test the hypotheses that dysfunctional transport of K+ across the BBB and/or attenuated volume changes and RBC deformability in response to elevated [K+]e and PO2 can explain the deficit in capillary hyperemia with hypertension and aging. This proposal is based on a long-term collaboration between two research groups with complementary in vivo and ex vivo expertise. The studies involve 2-photon imaging of CBF in vivo concurrently with recordings of [K+]e, PO2, and neural activity, as well as new ex vitro approaches, including microfluidic chambers, to study regulation of RBC flow kinetics in artificial capillaries devoid of the neurovascular unit. Together, the proposed studies represent an entirely novel approach to resolve the mechanisms of neurovascular coupling and define the effect of hypertension and aging on capillary flow, with direct comparison of cortical and subcortical white matter. Our hope is that these studies will identify novel therapeutic targets to interrupt or delay white matter loss arising from hypertension and aging.